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Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Harbin, 150030, Heilongjiang, China
Lactobacillus acidophilus LA-5 is a suitable probiotic for food application, but because of its slow growth in milk, an increase in its efficiency is desired. To shorten the time required for fermentation, the nutrient requirements of L. acidophilus LA-5 were analyzed, including the patterns of consumption of amino acids, purines, pyrimidines, vitamins, and metal ions. The nutrients required by L. acidophilus LA-5 were Asn, Asp, Cys, Leu, Met, riboflavin, guanine, uracil, and Mn2+, and when they were added to milk, the fermentation time of fermented milk prepared by L. acidophilus LA-5 alone was shortened by 9 h, with high viable cell counts that were maintained during storage of nutrient-supplemented fermented milk compared with the control. For fermented milk prepared by fermentation with Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, and L. acidophilus LA-5, viable cell counts of L. acidophilus LA-5 increased 1.3-fold and were maintained during storage of nutrient-supplemented fermented milk compared with the control. Adding nutrients had no negative effect on the quality of the fermented milk. The results indicated that suitable nutrients enhanced the growth of L. acidophilus LA-5 and increased its viable cell counts in fermented milk prepared by L. acidophilus LA-5 alone and mixed starter culture, respectively.
Lactobacillus acidophilus is a commercial strain and probiotic that is widely used in the dairy industry to obtain high-quality fermentation products (
Quality parameters of probiotic yogurt added to glucose oxidase compared to commercial products through microbiological, physical–chemical and metabolic activity analyses.
). Lactobacillus acidophilus LA-5 has the ability to reduce the serum cholesterol level, balance and stabilize the enteric microbiota, stimulate an immune response, improve lactose digestion, and potentially kill cancer cells (
). Fermented milk containing L. acidophilus LA-5 can protect against intestinal diseases by increasing beneficial bacteria and reducing potentially pathogenic bacteria (
). Moreover, the L. acidophilus LA-5 present in fermented milk was effective in reducing the Streptococcus mutans levels in saliva, and can also decrease the risk factors for acquiring nonalcoholic fatty liver disease (
Effect of short-term consumption of Amul probiotic yogurt containing Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 on salivary Streptococcus mutans count in high caries risk individuals.
Int. J. Appl. Basic Med. Res.2018; 8 (29744324): 111-115
). Accordingly, fermented milk is an ideal carrier of L. acidophilus LA-5.
Many fermented dairy products on the Chinese market have been manufactured with a single probiotic strain such as Yakult (Lactobacillus casei Shirota), LAJOIE (Lactobacillus paracasei LPC-37), and Cherita (Lactobacillus rhamnosus GG ATCC 53103). However, the fermentation time required to manufacture fermented milk with a single probiotic strain is long (
Growth and viability of Lactobacillus acidophilus NRRL B-4495, Lactobacillus casei NRRL B-1922 and Lactobacillus plantarum NRRL B-4496 in milk supplemented with cysteine, ascorbic acid and tocopherols.
). Quick production of fermented milk using L. acidophilus cannot occur because it also grows slowly in milk, which is unfavorable for the commercial development of fermented milk manufactured with a single probiotic strain. Therefore, to shorten the time required to produce fermented milk, preparation solely using L. acidophilus LA-5 should be considered. The survival of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus during transit through the human digestive environment remained controversial (
In vivo study of the survival of Lactobacillus delbrueckii ssp. bulgaricus CECT 4005T and Streptococcus thermophilus CECT 801 by DVC-FISH after consumption of fermented milk.
). Probiotics are often added to carry out co-fermentation with S. thermophilus and L. delbrueckii ssp. bulgaricus, including L. acidophilus; however, viable probiotic cell counts are often low in fermented milk (
). Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus grow faster than L. acidophilus during fermentation, and because L. delbrueckii ssp. bulgaricus produces lactic acid during fermentation and refrigerated storage, postacidification that causes loss of viability of L. acidophilus is an important factor to consider (
). Hence, increasing the viable cell counts and viability of L. acidophilus in fermented milk prepared by mixed starter culture should be considered.
Lactobacillus acidophilus is a fastidious organism with complex nutrient requirements. Its growth requires a variety of nutrients, including AA, vitamins, metal ions, buffers, and other compounds (
). It has been observed that milk lacking certain nutrients cannot support the rapid growth of L. acidophilus. The viability of L. acidophilus was improved in fermented milk supplemented with ascorbic acid and cysteine (
). The aforementioned studies showed that suitable nutrients enhanced the growth and viability of L. acidophilus. However, few studies have investigated the nutrient requirements of L. acidophilus LA-5 and its ability to ferment milk alone and ferment milk in a mixed starter culture for the purpose of shortening the fermentation time and increasing its viable cell counts and viability.
The purpose of the present study is to determine the optimal method for reducing the fermentation time of milk prepared solely by L. acidophilus LA-5 and increase its viable cell counts and viability in fermented milk prepared by a combination of S. thermophilus, L. delbrueckii ssp. bulgaricus, and L. acidophilus LA-5, based on the nutritional requirements of L. acidophilus LA-5.
MATERIALS AND METHODS
Strain, Culture Conditions, and Fermentation Experiments
Lactobacillus acidophilus LA-5, Streptococcus thermophilus CHCC 6483, and Lactobacillus delbrueckii ssp. bulgaricus-12 were obtained from Chr. Hansen (H⊘rsholm, Denmark). Lactobacillus acidophilus LA-5 was activated and subcultured in de Man, Rogosa, and Sharpe (MRS) broth (HuanKai Microbial Sci. and Tech. Co. Ltd., Guangdong, China) at 37°C. Cells were harvested by centrifugation (8,000 × g, 5 min, 4°C) and washed twice with PBS buffer (pH 7.2). Then, the culture was fermented in a 10-L Biotech-7000 bioreactor (Shanghai Baoxing, Shanghai, China) containing 7 L of chemically defined medium (CDM) with 2% (vol/vol) inoculum. The CDM was prepared according to
. The temperature and rotation speed were set at 37°C and 150 rpm, respectively. The growth rate was measured by spectrophotometric measurement at 600 nm. The cells were harvested at 0, 2.0, 4.5, 6.5, and 8.0 h by centrifugation (8,000 × g, 5 min, 4°C). The pellet and supernatant were stored at −80°C for the next analysis. Batch fermentations were independently repeated 3 times.
Effects of Nutrients on the Growth of L. acidophilus LA-5
The relationship of CDM components and the growth of L. acidophilus LA-5 has been described by the single nutrient omission method (
). Briefly, the nutrient was considered to be essential if its omission resulted in growth rates of less than half the maximum growth rate in the complete CDM. The nutrient was considered to be stimulatory when in its omission, the growth rate was between 50% and 80% of that observed in complete CDM. The nutrient was considered to be nonessential when in its omission, the growth rate was 80% (or more) of that obtained in the complete CDM.
Selection of Nutrients
Based on nutrient requirements, nutrients with a high consumption rate, AA with a high necessary rate, and essential and stimulatory nutrients were added to milk to conduct the preliminary single addition experiments with fermented milk. The aim of the preliminary single addition experiments was to select the nutrients that shorten the time required to prepare fermented milk using L. acidophilus LA-5. Accordingly, Asn, Asp, Leu, Met, riboflavin, guanine, uracil, and Mn2+ were selected, and the concentrations of these nutrients that were used are shown in Table 1.
Table 1Nutrients added to milk to assess effects on fermentation
Analysis of AA, Vitamins, Purines, Pyrimidines, and Ions
The whole-cell AA composition and the amounts of each AA that were consumed were determined by an amino acid analyzer (Acquity UPLC, Waters Corp., Milford, MA). Thiamine, riboflavin, nicotinic acid, Ca-pantothenate, pyridoxal, folic acid, cyanocobalamin, biotin, and inositol were measured by RP-HPLC with an UV detector (
Separation of water-soluble vitamins by reversed-phase high performance liquid chromatography with ultra-violet detection: Application to polyvitaminated premixes.
Simultaneous quantification of purine and pyrimidine bases, nucleosides and their degradation products in bovine blood plasma by high performance liquid chromatography tandem mass spectrometry.
The culture media MRS (for L. acidophilus and L. delbrueckii ssp. bulgaricus) and M17 (for S. thermophilus) were used to activate strains at 37°C. Strains were inoculated and propagated 2 times in MRS and M17 until strain activity was stable. Mother culture was prepared with sterilized skim milk that had 5% inoculation amount of the activated strains. The mother cultures of S. thermophilus and L. delbrueckii ssp. bulgaricus were cultivated at 43°C, and L. acidophilus was cultivated at 37°C. They were then stored at 4°C until the curd was firm. For fermented milk prepared using only L. acidophilus LA-5, milk was heated to 95°C for 5 min and cooled to 37°C, and then inoculated with 3% (vol/vol) of the mother culture of L. acidophilus LA-5 (with viable cell counts 2.0 × 106 cfu/mL) and incubated at 37°C until the pH of the milk reached 4.5. For fermented milk prepared using mixed starter culture, milk was heated to 95°C for 5 min and cooled to 43°C, and then inoculated with 3% (vol/vol) of mother culture of S. thermophilus, L. delbrueckii ssp. bulgaricus, and L. acidophilus LA-5 in a 1:1:1 ratio (with viable cell counts 2.0 × 106 cfu/mL for each strain), and incubated at 43°C until the pH of the milk reached 4.5. When fermentation was completed, all samples were quickly cooled in an ice bath and then stored at 4°C.
According to the nutrients selected, fermented milk was prepared with Asn, Asp, Cys, Leu, Met, riboflavin, guanine, uracil, and Mn2+ (Table 1). Fermented milk that had not been supplemented with nutrients was used as a control. Batch fermentations were repeated 3 independent times.
Microbiological, Physicochemical, Sensory, and Textural Analyses of Fermented Milk
Lactobacillus acidophilus LA-5, Streptococcus thermophilus CHCC 6483, and Lactobacillus delbrueckii ssp. bulgaricus-12 were enumerated in MRS-maltose agar (pH 6.4), M17 agar (pH 7.0), and MRS-glucose agar (pH 4.58), respectively (
Selective and differential enumerations of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt—A review.
Int. J. Food Microbiol.2011; 149 (21807435): 194-208
). Sample pH values were measured at room temperature with a combined glass electrode attached to a Jenway 3510 pH meter (Keison Products, Chelmsford, UK). The titratable acidity and water-retaining capability were determined according to
Processing and sensory characteristics of a fermented low-fat skim milk drink containing bioactive antihypertensive peptides, a functional milk product.
. Sensory properties of the fermented milk were assessed by 20 trained panelists recruited from staff members and students from the Northeast Agricultural University. Samples in 30-mL white plastic cups coded with 1 digit at room temperature were presented to each panelist. Water and crackers were given to panelists for palate cleansing between samples, allowing 15-min breaks between sessions. Panelists were advised not to swallow the product. Each panelist evaluated 4 samples for flavor, bitterness, texture, appearance, and acceptance, using a 10-point hedonic scale (1 = dislike extremely to 10 = like extremely).
Statistical Analysis
The data were analyzed with ANOVA using Statistica 9.2 software (StatSoft Inc., Tulsa, OK). The comparison between means was determined by using the Tukey's significant difference test (P < 0.05). All data were presented as the mean or mean ± standard deviation.
RESULTS
Nutrient Consumption Profiles
As shown in Figure 1a-c, the AA consumed in the greatest amounts were Asp + Asn (0.94 mmol/L), followed by Glu + Gln (0.69 mmol/L), Ala (0.55 mmol/L), and Leu (0.48 mmol/L); the AA consumed in the smallest amount was Phe (0.03 mmol/L). The remainder of the consumption amounts ranged from 0.06 to 0.44 mmol/L. For the consumption rate of AA, the highest rate was for Asp + Asn (88.97%), followed by Lys (81.98%), Leu (77.24%), Tyr (69.69%), Thr (68.94%), Glu + Gln (66.99%), and Ala (66.85%). The lowest consumption rate was for Phe (17.54%), and the remainder of the consumption rates ranged from 22.63 to 56.45%.
Figure 1Residual concentrations of (a, b, c) AA, (d) purines and pyrimidines, (e, f) vitamins, and (g, h) minerals at different culture times (t0 = 0 h, t1 = 2.0 h, t2 = 4.5 h, t3 = 6.5 h, t4 = 8.0 h). (a) The growth curve of Lactobacillus acidophilus LA-5. The results are expressed as the mean ± SD of 3 independent experiments. OD600 = optical density at 600 nm.
As shown in Figure 1d, for purines and pyrimidines, the largest consumption amount was for uracil (0.0463 mmol/L), followed by adenine (0.0341 mmol/L) and thymine (0.0336 mmol/L). The smallest consumption amount was for xanthine (0.0182 mmol/L), followed by guanine (0.0301 mmol/L). The highest and lowest consumption rates were uracil at 65.90% and xanthine at 40.85%. The consumption rates of guanine, thymine, and adenine were 53.15%, 50.05%, and 49.94%, respectively.
As shown in Figure 1e-f, the largest consumption amount of vitamins was for p-aminobenzoic acid (0.0085 mmol/L), followed by biotin (0.0060 mmol/L), inositol (0.0043 mmol/L), and pyridoxal (0.0021 mmol/L). The least consumed vitamin was cyanocobalamin (0.00008 mmol/L). The remainder of the consumption amounts of vitamins ranged from 0.0003 to 0.0013 mmol/L. The highest consumption rate was for riboflavin (61.70%), followed by thiamine (29.46%), Ca-pantothenate (25.96%), and folic acid (19.61%). The lowest consumption rate was for p-aminobenzoic acid (14.90%). The remainder of the consumption rates of vitamins ranged from 17.48 to 19.52%.
As shown in Figure 1g-h, the largest amount of minerals consumed was for Na+ (10.36 mmol/L), with a consumption rate of 18.90%, followed by K+ (4.22 mmol/L) and Mg2+ (0.531 mmol/L) at consumption rates of 16.07% and 38.32%, respectively. The consumption of Mn2+ was 0.141 mmol/L, but its consumption rate was the highest (91.02%). The least consumed ion was Fe2+ (0.0354 mmol/L) at a consumption rate of 58.28%.
Necessary AA Profiles
According to the concentrations of AA in the whole-cell hydrolysates, the required amounts of AA were analyzed. The ratio of the requirement of necessary AA during different periods to all required amounts of AA in the fermentation process is presented in Figure 2. The proportion of Glu + Gln was 10.71%, and was higher than the amount of other AA demanded for the growth of L. acidophilus LA-5. The Ala (9.73%), Arg (9.71%), and Asp + Asn (8.70%) were next, followed by Pro (7.19%), Leu (6.68%), and Lys (6.28%). The required amounts of Met, Cys, Phe, Val, Tyr, Ile, Gly, Ser, His, and Thr ranged from 2.59 to 5.20% of all AA required.
Figure 2Necessary AA at different culture times (t1 = 2.0 h, t2 = 4.5 h, t3 = 6.5 h, t4 = 8.0 h). The results are expressed as the mean ± SD of 3 independent experiments.
Effects of Nutrients on the Growth of L. acidophilus LA-5
As Figure 3 shows, Cys, Glu, Ile, Leu, Lys, Met, Phe, Thr, Try, Val, Arg, and glucose are essential nutrients for the growth of L. acidophilus LA-5. The Gly, Ca-pantothenate, and Mn2+ were stimulatory nutrients that stimulated the growth of L. acidophilus LA-5. Others were nonessential nutrients that had no obvious effects on the growth of L. acidophilus LA-5.
Figure 3Effect of nutrient omission on the growth rate of Lactobacillus acidophilus LA-5. The growth rate is expressed as a percentage and is based on the growth rate when a nutrient was omitted from the chemically defined medium (CDM) compared with that in the CDM. The results are expressed as the mean ± SD of 3 independent experiments.
According to the present results, some nutrients that are lacking in milk were necessary for L. acidophilus LA-5 (Table 1). Based on the consumption patterns of nutrients and their concentrations in milk, AA with higher consumption rates (more than 50%) should be added to milk, whereas the consumption rates of purines, pyrimidine, vitamins, and ions were greater than 20%. Therefore, Asp, Asn, Lys, Leu, Tyr, Thr, Glu, Gln, Ala, Ile, guanine, adenine, xanthine, uracil, thymine, thiamine, riboflavin, Ca-pantothenate, Mg2+, Fe2+, and Mn2+ were supplied to milk for the preliminary single addition experiments. According to the necessary AA profiles and their concentrations in milk, AA with higher requirement ratios (close to or more than 8%) should also be added, and therefore, Arg and Pro were added to milk. In addition, essential nutrients and growth-stimulating nutrients, namely, Cys, Met, Phe, Val, and Gly, were also added for the preliminary single-addition experiments. Figure 4a shows the nutrients that promoted the fermentation process. The fermentation time in single nutrient-supplemented fermented milk was shortened by at least 2 h compared with that of the control sample. According to the results, Asn, Asp, Cys, Leu, Met, riboflavin, guanine, uracil, and Mn2+ were added to milk together, and concentrations of these nutrients are shown in Table 1.
Figure 4(a) Changes in the pH of samples after nutrients were added that promoted fermentation by Lactobacillus acidophilus LA-5, and (b) the time it takes for L. acidophilus LA-5 to ferment nutrient-added milk. Formulation represents milk that was supplemented with 9 selected nutrients. (c) Changes in the viable cell counts of L. acidophilus LA-5 in control and nutrient-supplemented fermented milk prepared solely by L. acidophilus LA-5 during storage at 4°C. (d, e, f) Changes in the viable cell counts of L. acidophilus LA-5, Streptococcus thermophilus, and Lactobacillus delbrueckii ssp. bulgaricus in control and nutrient-supplemented fermented milk prepared by mixed starter culture during storage at 4°C, respectively. The results are expressed as the mean or the mean ± SD of 3 independent experiments. Symbols with different letters differ significantly (P < 0.05).
Effects of Nutrients on the Properties of Fermented Milk
When fermented milk was prepared solely by L. acidophilus LA-5, compared with the control, the fermentation time of the milk supplemented with the nutrients was reduced approximately 9 h (Figure 4a-b). The viable cell counts of L. acidophilus LA-5 in fermented milk prepared solely by L. acidophilus LA-5 during storage are shown in Figure 4c. At the end of the fermentation process, the viable cell counts of fermented milk supplemented with the nutrients reached 2.17 × 109 cfu/mL, which was approximately 1.8-fold that of the control. Nutrient-supplemented fermented milk prepared solely by L. acidophilus LA-5 contained significantly higher levels of L. acidophilus LA-5 than the control sample (P < 0.05). Hence, the nutrients had positive effects on the growth and maintenance of L. acidophilus LA-5 viability. At the end of storage, the pH value decreased by 0.4 to 0.5 units, and similar trends in pH decline were observed in all of the samples (Figure 5a). For fermented milk prepared by mixed starter culture, there were no significant differences in fermentation time (approximately 5 h) between the control and nutrient-supplemented fermented milk, and the pH value decreased by 0.3 to 0.4 units during storage (Figure 5a-b). The viable cell counts of L. acidophilus LA-5 in fermented milk prepared by mixed starter culture during storage are shown in Figure 4d. Nutrient-supplemented fermented milk prepared by mixed starter culture contained significantly higher levels of L. acidophilus LA-5 than the control sample (P < 0.05), the viable cell counts of L. acidophilus LA-5 in nutrient-supplemented fermented milk prepared by mixed starter culture were approximately 2.3-fold that of the control when fermentation was completed. At the end of the storage period, the survival of L. acidophilus LA-5 in the nutrient-supplemented fermented milk prepared by mixed starter culture was 2-fold that of the control. However, there was not a significant difference (P > 0.05) in the viable cell counts of S. thermophilus and L. delbrueckii ssp. bulgaricus (Figure 4e-f). As Figure 5c, Table 2, Figure 5d, and Table 3 show, no differences were observed in the textural and sensory properties between the control and nutrient-supplemented fermented milk.
Figure 5(a) Changes in the pH of fermented milk prepared solely by Lactobacillus acidophilus LA-5 and fermented milk prepared by mixed starter culture samples during storage at 4°C. ●, ■ = fermented milk and control, respectively, prepared solely with L. acidophilus LA-5; ▼, ▲ = fermented milk and control prepared with mixed starter culture. (b) Changes in the pH of fermented milk prepared by mixed starter culture during fermentation. (c) Graphic representation of the mean sensory evaluation by quantitative descriptive analysis of control and nutrient-supplemented fermented milk prepared solely by L. acidophilus LA-5. (d) Graphic representation of the mean sensory evaluation by quantitative descriptive analysis of control and nutrient-supplemented fermented milk prepared by mixed starter culture. The results are expressed as the mean or the mean ± SD of 3 independent experiments.
). In the current study, small select nutrients enhanced the growth of L. acidophilus LA-5 in milk. Accordingly, improving the nutrient-limiting conditions might be an effective way to accelerate the fermentation process. Every species of Lactobacillus has distinctive growth requirements for essential energy, and carbon and nitrogen sources (
). However, the ability of Lactobacillus to synthesize some nutrients such as AA is limited. Therefore, an exogenous source of nutrients is required for the most optimal growth (
). Because L. acidophilus LA-5 is an important commercial starter strain, it is necessary to determine its nutrient requirements so that it can be efficiently utilized to ferment specific foods. The present results indicated that nutrients with a high consumption rate, AA with a high necessary rate, and growth-stimulating and essential nutrients should be considered in specific fermented foods manufactured with L. acidophilus LA-5 so that its viable cell counts are increased and fermentation time is shortened.
The Asn, Asp, Cys, Leu, Met, riboflavin, guanine, uracil, and Mn2+ reduced the fermentation time and increased viable cell counts of L. acidophilus LA-5 in fermented milk. The genome of L. acidophilus NCFM is closely related to L. acidophilus LA-5, and the results showed that L. acidophilus has the ability to synthesize Cys, Asp, Asn, Met, and guanine but not Leu, riboflavin, or uracil (
). The Cys, Asp, and guanine were synthesized through a de novo pathway, Met was synthesized by Ser and Cys, Asn could be produced via Asp, and all involved complicated synthesis pathways. Additionally, L. acidophilus was auxotrophic for guanine and uracil because they could not reduce ribonucleotide to the corresponding deoxyribonucleotide for DNA synthesis (
). The supplied substrates for synthetizing nutrients were deficient in milk, or the rates of synthesis of these nutrients could not meet the growth requirements of L. acidophilus LA-5. Additionally, L. acidophilus LA-5 obtained AA by hydrolyzing milk protein, which did not support good growth. Hence, these nutrients were important to enhance the growth of L. acidophilus LA-5 in milk. If the supplied nutrients were absent or insufficient, the high level of viable cell counts and high survival rate of L. acidophilus LA-5 were not achieved.
The Cys increased the viable cell counts of L. acidophilus in milk by acting as one of the main redox potential donors, and increasing the redox potential during storage (
Growth and viability of Lactobacillus acidophilus NRRL B-4495, Lactobacillus casei NRRL B-1922 and Lactobacillus plantarum NRRL B-4496 in milk supplemented with cysteine, ascorbic acid and tocopherols.
Growth kinetics and lactic acid production of Lactobacillus plantarum NRRL B-4496, L. acidophilus NRRL B-4495, and L. reuteri B-14171 in media containing egg white hydrolysates.
, Asp and Leu were the main free AA present in egg white hydrolysates, higher yields of cell biomass and production of lactic acid was observed in L. acidophilus NRRL B-4495 when they grew in a medium containing egg white hydrolysates. The Leu enhanced the growth of L. acidophilus, which was essential for L. acidophilus throughout the fermentation period in both ovine and milk (
). Guanine significantly stimulated the growth of L. johnsonii NCC 533, a member of the acidophilus group of intestinal lactobacilli, in whole and skim milk, which may be due to it serving as a deoxyribonucleotide substrate for bacterial growth (
). For L. acidophilus LA-5, the physiological functions and requirements of these nutrients may be the main reasons for promoting the fermentation of fermented milk. The result indicated that they are likely to promote the metabolism and growth of L. acidophilus LA-5 as a stimulating factor.
The levels of dissolved oxygen, hydrogen peroxide, and storage conditions could result in reduced viability of probiotics (
). Generally, L. acidophilus does not possess a sufficient scavenging mechanism, and therefore, the intracellular accumulation of toxic oxygenic metabolites such as superoxide anion and hydroxyl radical could lead to its death (
). Onions are a source of sulfur compounds, vitamins, and minerals, from which these compounds might scavenge hydrogen peroxide during the fermentation of L. acidophilus NCFM (
, Mn+-metabolite complexes could protect essential enzymes from oxidative damage. For L. acidophilus LA-5, maintaining high viable cell counts and viability might be attributed to additional nutrients that are able to scavenge hydrogen peroxide and protect L. acidophilus during storage of fermented milk. The nutrient supplementation did not affect the viable cell counts of S. thermophilus and L. delbrueckii ssp. bulgaricus in this study. It has been observed that the growth of S. thermophilus and L. delbrueckii ssp. bulgaricus was suppressed when 250 or 500 mg/L of Cys was supplied (
The present results indicate that suitable concentrations of available nutrients in fermented milk prepared by mixed starter culture were necessary to promote the growth of L. acidophilus LA-5 without affecting the growth of other strains. Additionally, no obvious difference was observed in the quality between the control and nutrient-supplemented fermented milk, which was consistent with similar results obtained by
Production costs of fermented milk mainly includes the cost of raw milk, cost of auxiliary materials, costs of fuel and power, labor costs, undepreciated cost of the asset, management cost, and packing cost. The present results showed that the fermentation time of fermented milk prepared solely by L. acidophilus LA-5 was shortened by 9 h by addition of nutrients. Compared with the production costs of fermented milk supplemented without the nutrients, cost of auxiliary materials increases 0.48%, and fuel and power costs, labor costs, undepreciated cost of the asset, and management cost decrease 34.4% in fermented milk supplemented with the nutrients. Detailed information about the formula for the cost of the proposed technology can be found in Supplemental File S1 (https://doi.org/10.3168/jds.2020-18953;
Workshop scheduling using practical (inaccurate) data Part 2: An investigation of the robustness of scheduling rules in a dynamic and stochastic environment.
Supplementation with Asn, Asp, Cys, Leu, Met, riboflavin, guanine, uracil, and Mn2+ shortened the fermentation time of milk by 9 h. The viable cell counts and viability of L. acidophilus LA-5 were approximately 1.8-fold and 1.4-fold that of the control, respectively, in fermented milk prepared only with L. acidophilus LA-5. Furthermore, the viable cell counts and viability of L. acidophilus LA-5 were approximately 2.3-fold and 2-fold that of the control, respectively, in nutrient-supplemented fermented milk prepared by mixed starter culture containing L. acidophilus LA-5. The present research provides a reference model for regulating the growth of probiotics in fermented milk prepared by a single probiotic strain and in fermented milk prepared by a mixed starter culture containing the probiotic strain, based on the nutrient requirements of probiotics.
ACKNOWLEDGMENTS
This work was supported by grants from the National Key Research and Development Plan Project of China (2018YFD0400405), National Natural Science Foundation of China (31771989). It should be understood that none of the authors have any financial or scientific conflict of interest with regard to the research described in this manuscript.
Processing and sensory characteristics of a fermented low-fat skim milk drink containing bioactive antihypertensive peptides, a functional milk product.
Selective and differential enumerations of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt—A review.
Int. J. Food Microbiol.2011; 149 (21807435): 194-208
Effect of short-term consumption of Amul probiotic yogurt containing Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 on salivary Streptococcus mutans count in high caries risk individuals.
Int. J. Appl. Basic Med. Res.2018; 8 (29744324): 111-115
Quality parameters of probiotic yogurt added to glucose oxidase compared to commercial products through microbiological, physical–chemical and metabolic activity analyses.
In vivo study of the survival of Lactobacillus delbrueckii ssp. bulgaricus CECT 4005T and Streptococcus thermophilus CECT 801 by DVC-FISH after consumption of fermented milk.
Separation of water-soluble vitamins by reversed-phase high performance liquid chromatography with ultra-violet detection: Application to polyvitaminated premixes.
Workshop scheduling using practical (inaccurate) data Part 2: An investigation of the robustness of scheduling rules in a dynamic and stochastic environment.
Growth kinetics and lactic acid production of Lactobacillus plantarum NRRL B-4496, L. acidophilus NRRL B-4495, and L. reuteri B-14171 in media containing egg white hydrolysates.
Growth and viability of Lactobacillus acidophilus NRRL B-4495, Lactobacillus casei NRRL B-1922 and Lactobacillus plantarum NRRL B-4496 in milk supplemented with cysteine, ascorbic acid and tocopherols.
Simultaneous quantification of purine and pyrimidine bases, nucleosides and their degradation products in bovine blood plasma by high performance liquid chromatography tandem mass spectrometry.
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